The pyrethroids are one of the most heavily used insecticide classes in the world. Sensitive and rapid analytical techniques are needed for assessments of human exposure to these compounds. Highly sensitive and selective ELISAs for glycine conjugates of esfenvalerate key metabolites phenoxybenzoic acid (PBA) and s-fenvalerate acid (sFA) were developed. Rabbits were immunized with either N-(3-phenoxybenzoyl)-4-amino-L-phenylalanine-fetuin or N-[(S)-4-chloro-2-(methylethyl)benzeneacetyl]-4-amino-L-phenyla lan ine -fetuin, and all sera were screened against numerous coating antigens. The antibodies with the least interference and best sensitivity were optimized and characterized. The I(50)s for sFA-glycine and PBA-glycine in buffer were found to be 0.40 +/- 0.12 microg/L (1.47 +/- 0.44 nmol/L) and 0.42 +/- 0.18 microg/L (1.56 +/- 0.67 nmol/L), respectively. Both assays exhibited high selectivity. Little or no cross reactivity to the parent compound and other metabolites was measured. The matrix effects of urine were investigated. Solid-phase extraction (SPE) strategies were used in an attempt to decrease the matrix effects and increase the sensitivity of the overall method. The limit of quantitation (LOQ) for both sFA-glycine and PBA-glycine in urine with SPE is 1.0 microg/L (3.70 nmol/L). These assays could be used as markers of exposure for monitoring biological samples.